The p53 tumor suppressor gene is frequently inactivated by mutations in human cancers. p53 is a sequence-specific transcription factor, whose activity is regulated by DNA damage, and activates expression of genes that induce cell cycle arrest or apoptosis. Regulation of p53 itself is complex and subject to DNA damage-regulated posttranslational modifications. These modifications include acetylation and phosphorylation. In the previous grant period our studies uncovered novel pathways for regulation of p53 via modifications. We demonstrated that methylation and demethylation at lysine 370 (K370) are involved in regulating p53 in response to DNA damage. Our preliminary data show that a novel site, K373, is also methylated. Based on these observations, and methylation at additional p53 residues detected by others, we hypothesize that methylation of p53 serves to regulate p53 positively and negatively, and cross-talks with phosphorylation and acetylation. We also uncovered an unanticipated novel pathway in the nucleus, where LKB1, the Peutz=Jeager kinase, and its downstream target, AMPK, function as transcriptional coactivators for p53. The kinases are directly recruited to p53-regulated promoters and respond to multiple cellular stress pathways, including both DNA damage and metabolic stress. AMPK directly phosphorylates p53, and also phosphorylates a chromatin target, histone H2B. Based on these observations we propose that many enzymes carry out post-translational modifications of both factors, such as p53, and chromatin. For example, the serine/threonine kinase AMPK may function to target both p53 and histones in a coordinated fashion, and this may also be the case for lysine methyltransferases. In general, this coordination may lead to interrelated factor/histone modifications that reinforce one another in activating or repressing transcription. Our studies will focus on the following specific aims: (1) we will investigate multiple methylation states of p53 in negative and positive regulation of p53 activity, including a novel methylation site at K373 carried out by the G9a and Glp methylases. (2) We will examine p53 phosphorylation by AMPK kinase, and mechanisms of recruitment of LKB1/AMPK protein complexes to promoters. (3) We will investigate the role of LKB1/AMPK, and the orthologous Snf1 kinase, in histone H2B phosphorylation. The proposed studies will advance our understanding of mechanisms by which p53 activates gene expression in response to cellular stress, raising the likelihood for pharmacologic regulation of p53 function in human cancer (p53 methylation), diabetes and obesity (p53 phosphorylation) in the future.